The genus Clostridium is comprised of gram-positive, anaerobic, spore-forming bacilli. The natural habitat of these organisms is the environment and the intestinal tracts of humans and other animals. Indeed, clostridia are ubiquitous; they are commonly found in soil, dust, sewage, marine sediments, decaying vegetation, and mud. (See e.g., Sneath et al., "Clostridium," Bergey's Manual.RTM. of Systematic Bacteriology, Vol. 2, pp. 1141-1200, Williams & Wilkins [1986]). Despite the identification of approximately 100 species of Clostridium, only a small number have been recognized as etiologic agents of medical and veterinary importance. Nonetheless, these species are associated with very serious diseases, including botulism, tetanus, anaerobic cellulitis, gas gangrene, bacteremia, pseudomembranous colitis, and clostridial gastroenteritis. Table 1 lists some of the species of medical and veterinary importance and the diseases with which they are associated. As virtually all of these species have been isolated from fecal samples of apparently healthy persons, some of these isolates may be transient, rather than permanent residents of the colonic flora.
TABLE 1 Clostridium Species Of Medical And Veterinary Importance* Species Disease C. aminovalericum Bacteriuria (pregnant women) C. argentinese Infected wounds; Bacteremia; Botulism; Infections of amniotic fluid C. baratii Infected war wounds; Peritonitis; Infectious processes of the eye, ear and prostate C. beijerinckikii Infected wounds C. bifermentans Infected wounds; Abscesses; Gas Gangrene; Bacteremia C. botulinum Food poisoning; Botulism (wound, food, infant) C. butyricum Urinary tract, lower respiratory tract, pleural cavity, and abdominal infections; Infected wounds; Abscesses; Bacteremia C. cadaveris Abscesses; Infected wounds C. carnis Soft tissue infections; Bacteremia C. chauvoei Blackleg C. clostridioforme Abdominal, cervical, scrotal, pleural, and other infections; Septicemia; Peritonitis; Appendicitis C. cochlearium Isolated from human disease processes, but role in disease unknown. C. difficile Antimicrobial-associated diarrhea; Pseudomembranous enterocolitis; Bacteremia; Pyogenic infections C. fallax Soft tissue infections C. ghnoii Soft tissue infections C. glycolicum Wound infections; Abscesses; Peritonitis C. hastiforme Infected war wounds; Bacteremia; Abscesses C. histolyticum Infected war wounds; Gas gangrene; Gingival plaque isolate C. indolis Gastrointestinal tract infections C. innocuum Gastrointestinal tract infections; Empyema C. irregulare Penile lesions C. leptum Isolated from human disease processes, but role in disease unknown. C. limosum Bacteremia; Peritonitis; Pulmonary infections C. malenominatum Various infectious processes C. novyi Infected wounds; Gas gangrene; Blackleg, Big head (ovine); Redwater disease (bovine) C. oroticum Urinary tract infections; Rectal abscesses. C. paraputrificum Bacteremia; Peritonitis; Infected wounds; Appendicitis C. perfringens Gas gangrene; Anaerobic cellulitis; Intra-abdominal abscesses; Soft tissue infections; Food poisoning; Necrotizing pneumonia; Empyema; Meningitis; Bacteremia; Uterine Infections; Enteritis necrotans; Lamb dysentery; Struck; Ovine Enterotoxemia; C. putrefaciens Bacteriuria (Pregnant women with bacteremia) C. putrificum Abscesses; Infected wounds; Bacteremia C. ramosum Infections of the abdominal cavity, genital tract, lung, and biliary tract; Bacteremia C. sartagoforme Isolated from human disease processes, but role in disease unknown. C. septicum Gas gangrene; Bacteremia; Suppurative infections; Necrotizing enterocolitis; Braxy C. sordellii Gas gangrene; Wound infections; Penile lesions; Bacteremia; Abscesses; Abdominal and vaginal infections C. sphenoides Appendicitis; Bacteremia; Bone and soft tissue infections; Intraperitoneal infections; Infected war wounds; Visceral gas gangrene; Renal abscesses C. sporogenes Gas gangrene; Bacteremia; Endocarditis; central nervous system and pleuropulmonary infections; Penile lesions; Infected war wounds; Other pyogenic infections C. subterminale Bacteremia; Empyema; Biliary tract, soft tissue and bone infections C. symbiosum Liver abscesses; Bacteremia; Infections resulting due to bowel flora C. tertium Gas gangrene; Appendicitis; Brain abscesses; Intestinal tract and soft tissue infections; Infected war wounds; Periodontitis; Bacteremia C. tetani Tetanus; Infected gums and teeth; Corneal ulcerations; Mastoid and middle ear infections; Intraperitoneal infections; Tetanus neonatorum; Postpartum uterine infections; Soft tissue infections, especially related to trauma (including abrasions and lacerations); Infections related to use of contaminated needles C. thermosaccharolyticum Isolated from human disease processes, but role in disease unknown. *Compiled from Engelkirk et al. "Classification", Principles and Practice of Clinical Anaerobic Bacteriology, pp. 22-23, Star Publishing Co., Belmont, CA (1992); Stephen and Petrowski, "Toxins Which Traverse Membranes and Deregulate Cells," in Bacterial Toxins, 2d ed., pp. 66-67, American Society for Microbiology (1986); Berkow and Fletcher (eds.), "Bacterial Diseases," Merck Manual of Diagnosis and Therapy, 16th ed., pp. 116-126, Merck Research Laboratories, Rahway, N.J. (1992); and Siegmond and Fraser (eds.), "Clostridial Infections," Merck Veterinary Manual, 5th ed., pp. 396-409, Merck & Co., Rahway, N.J. (1979).
In most cases, the pathogenicity of these organisms is related to the release of powerful exotoxins or highly destructive enzymes. Indeed, several species of the genus Clostridium produce toxins and other enzymes of great medical and veterinary significance (Hatheway, Clin. Microbiol. Rev., 3:66-98 [1990]).
Perhaps because of their significance for human and veterinary medicine, much research has been conducted on these toxins, in particular those of C. botulinum, C. tetani, and C. peringens, although much recent work has also been conducted on C. difficile.
C. botulinum
Several strains of Clostridium botulinum produce toxins of significance to human and animal health (Hatheway, Clin. Microbiol. Rev., 3:66-98 (1990]). The effects of these toxins range from diarrheal diseases that can cause destruction of the colon, to paralytic effects that can cause death. Neonates and humans and animals in poor health (e.g, those suffering from diseases associated with old age or immunodeficiency diseases) are particularly at risk for developing severe clostridial diseases such as botulism.
Clostridium botulinum produces the most poisonous biological toxin known, with a lethal human dose in the nanogram range. Botulinal toxin blocks nerve transmission to the muscles, resulting in flaccid paralysis. When the toxin reaches airway and respiratory muscles, it results in respiratory failure that can cause death (Arnon, J. Infect. Dis., 154:201-206 [1986]).
C. botulinum spores are carried by dust and are found on vegetables taken from the soil, on fresh fruits, and on agricultural products such as honey. Under conditions favorable to the organism, the spores germinate to vegetative cells, resulting in the production of the toxin (Arnon, Ann. Rev. Med., 31:541 [1980]).
Botulism disease may be grouped into four types, based on the method of introduction of toxin into the bloodstream. Food-borne botulism results from ingesting improperly preserved and inadequately heated food that contains botulinal toxin (i.e., the toxin is pre-formed prior to ingestion). There were 355 cases of food-borne botulism in the United States between 1976 and 1984 (MacDonald et al., Am. J. Epidemiol., 124:794 [1986]). The death rate due to botulinal toxin has been reported as 12% and can be higher in particular risk groups (Tacket et al., Am. J. Med., 76:794 [1984]). Wound-induced botulism results from C. botulinum penetrating traumatized tissue and producing toxin that is absorbed into the bloodstream. Since 1950, thirty cases of wound botulism have been reported (Swartz, "Anaerobic Spore-Forming Bacilli: The Clostridia," pp. 633-646, in Davis et al., (eds.), Microbiology, 4th edition, J. B. Lippincott Co. [1990]). Inhalation botulism results when the toxin is inhaled. Inhalation botulism has been reported as the result of accidental exposure in the laboratory (Holzer, Med. Klin., 41:1735 [1962]) and is a potential danger if the toxin is used as an agent of biological warfare (Franz et al., in Botulinum and Tetanus Neurotoxins, DasGupta (ed.), Plenum Press, New York [1993], pp. 473-476). Infectious infant botulism results from C. botulinum colonization of the infant intestine with production of toxin and its absorption into the bloodstream. It is likely that the bacterium gains entry when spores are ingested and subsequently germinate (Arnon, J. Infect. Dis., 154:201 [1986]). There have been 500 cases reported since it was first recognized in 1976 (Swartz, supra.).
Infant botulism strikes infants who are three weeks to eleven months old (greater than 90% of the cases are infants less than six months) (Arnon, J. Infect. Dis., 154:201 [1986]). Clinical symptoms of infant botulism range from mild paralysis, to moderate and severe paralysis requiring hospitalization, to fulminant paralysis, leading to sudden death (Arnon, Epidemiol. Rev., 3:45 [1981]). It is believed that infants are susceptible, due, in large part, to the absence of the fill adult complement of intestinal microflora. The benign microflora present in the adult intestine provide an acidic environment that is not favorable to colonization by C. botulinum. In contrast, infants begin life with a sterile intestine which is gradually colonized by microflora. Because of the limited microflora present in early infancy, the intestinal environment is not as acidic, allowing for C. botulinum spore germination, growth, and toxin production. In this regard, some adults who have undergone antibiotic therapy which alters intestinal microflora become more susceptible to botulism. An additional factor accounting for infant susceptibility to infectious botulism is the immaturity of the infant immune system.
Infant botulism has been implicated as the cause of mortality in some cases of Sudden Infant Death Syndrome (SIDS, also known as crib death). SIDS is officially recognized as infant death that is sudden and unexpected and that remained unexplained despite complete post-mortem examination. The link of SIDS to infant botulism came when fecal or blood specimens taken at autopsy from SIDS infants were found to contain C. botulinum organisms and/or toxin in 3-4% of cases analyzed (Peterson et al., Rev. Infect. Dis., 1:630 [1979]). In contrast, only 1 of 160 healthy infants (0.6%) had C. botulinum organisms in the feces and no botulinal toxin (Arnon et al., Lancet, pp. 1273-76, Jun. 17, 1978.)
In developed countries, SIDS is the number one cause of death in children between one month and one year old (Arnon et al., Lancet, pp. 1273-77, Jun. 17, 1978.) More children die from SIDS in the first year than from any other single cause of death in the first fourteen years of life. In the United States, there are 8,000-10,000 SIDS victims annually (Id).
The chief therapy for severe infant botulism is ventilatory assistance using a mechanical respirator and concurrent elimination of toxin and bacteria using cathartics, enemas, and gastric lavage. There were 68 hospitalizations in California for infant botulism in a single year with a total cost of over $4 million for treatment (Frankovich and Arnon, West. J. Med., 154:103 [1991]).
Different strains of Clostridium botulinum each produce antigenically distinct toxin designated by the letters A-G. Serotype A toxin has been implicated in 26% of the cases of food botulism; types B, E and F have also been implicated in a smaller percentage of the food botulism cases (Sugiyama, Microbiol. Rev., 44:419 [1980]). Wound botulism has been reportedly caused by only types A or B toxins (Sugiyama, supra). Nearly all cases of infant botulism have been caused by bacteria producing either type A or type B toxin (exceptionally, one New Mexico case was caused by Clostridium botulinum producing type F toxin and another by Clostridium botulinum producing a type B-type F hybrid) (Arnon, Epidemiol. Rev., 3:45 [1981]). Type C toxin affects waterfowl, cattle, horses and mink. Type D toxin affects cattle, and type E toxin affects both humans and birds.
A trivalent antitoxin derived from horse plasma is commercially available from Connaught Industries Ltd. as a therapy for toxin types A, B, and E. However, the antitoxin has several disadvantages. First, extremely large dosages must be injected intravenously and/or intramuscularly. Second, the antitoxin has serious side effects such as acute anaphylaxis which can lead to death, and serum sickness. Finally, the efficacy of the antitoxin is uncertain and the treatment is costly (Tacket et al., Am. J. Med., 76:794 [1984]).
A heptavalent equine botulinal antitoxin which uses only the F(ab')2 portion of the antibody molecule has been tested by the United States Military (Balady, USAMRDC Newsletter, p. 6 [1991]). This was raised against impure toxoids in those large animals and is not a high titer preparation.
A pentavalent human antitoxin has been collected from immunized human subjects for use as a treatment for infant botulism. The supply of this antitoxin is limited and cannot be expected to meet the needs of all individuals stricken with botulism disease. In addition, collection of human sera must involve screening out HIV and other potentially serious human pathogens (Schwarz and Arnon, Western J. Med., 156:197 [1992]).
Immunization of subjects with toxin preparations has been done in an attempt to induce immunity against botulinal toxins. A C. botulinum vaccine comprising chemically inactivated (i.e., formaldehyde-treated) type A, B, C, D and E toxin is commercially available for human usage. However, this vaccine preparation has several disadvantages. First, the efficacy of this vaccine is variable (in particular, only 78% of recipients produce protective levels of anti-type B antibodies following administration of the primary series). Second, immunization is painful (deep subcutaneous inoculation is required for administration), with adverse reactions being common (moderate to severe local reactions occur in approximately 6% of recipients upon initial injection; this number rises to approximately 11% of individuals who receive booster injections) (Informational Brochure for the Pentavalent (ABCDE) Botulinum Toxoid, Centers for Disease Control). Third, preparation of the vaccine is dangerous as active toxin must be handled by laboratory workers.
What is needed are safe and effective vaccine preparations for administration to those at risk of exposure to C. botulinum toxins. More efficacious methods for treatment of botulism disease are also needed.
C. perfringens
C. perfringens is reported to be the most widely occurring pathogenic bacterium (See, Hatheway, supra, at p. 77). The organism, first described by Welch and Nuttall in 1892, and named Bacillus aerogenes capsulatus, has also been commonly referred to as C. welchii. C. perfringens is commonly isolated from soil samples, as well as the intestinal contents of humans and other animals. Although other Clostridium species are also associated with gas gangrene (e.g., C. novyi, C. septicum, C. histolyticum, C. tertium, C. bifermentans, and C. sporogenes), C. perfringens is the species most commonly involved. These organisms are not highly pathogenic when introduced into healthy tissue, but are associated with rapidly progressive, devastating infections characterized by the accumulation of gas and extensive muscle and tissue necrosis, when introduced in the presence of tissue injury (e.g., damaged muscle). During active multiplication, invasive strains of clostridia produce exotoxins with necrotizing (i.e., cytolytic), hemolytic, and/or lethal properties. In addition, enzymes such as collagenase proteinase, deoxyribonuclease, and hyaluronidase produced by the organisms result in the accumulation of toxic degradation products in the tissues.
C. perfringens produces four major lethal toxins (alpha, beta, epsilon, and iota), upon which the toxin types of the species are based, as well as nine minor toxins (or soluble antigens), that may or may not be involved in the pathogenicity associated with the organism (See, Hatheway, supra, at 77). These minor toxins are delta, theta, kappa, lambda, mu, nu, gamma, eta, and neuraminidase. In addition, some strains produce an enterotoxin that is responsible for C. perfringens food-borne disease. C. perfringens may be divided into "toxin types" designated as A, B, C, D, and E, based on the toxins produced. For example, most strains of toxin type A produce the alpha toxin, but not the other major lethal toxins (i.e., beta, epsilon, and iota); toxin type B organisms produce all of the major lethal toxins with the exception of iota toxin; toxin type C organisms produce alpha and beta major lethal toxins, but not epsilon or iota toxins; toxin type D organisms produce alpha and epsilon toxins, but not beta or iota toxins; and toxin type E organisms produce alpha and iota toxins, but not beta or epsilon toxins.
The alpha toxin is a lecithinase (phospholipase C), while the beta toxin is a necrotizing, trypsin-labile toxin, the epsilon toxin is a permease, trypsin-activatable toxin, and iota toxin is a dermonecrotic, binary, ADP-ribosylating, trypsin-activatable toxin. The delta toxin is a hemolysin, the theta toxin is an oxygen-labile hemolysin, and cytolysin, the kappa toxin is a collagenase and gelatinase, the lambda toxin is a protease, the mu toxin is a hyaluronidase, and the nu toxin is a DNase. The gamma and eta toxins have not been well-characterized and their existence is questionable (See, Hatheway, supra, at p. 77). The neuraminidase is an N-acetylneuraminic acid glycohydrolase, and the enterotoxin is enterotoxic and cytotoxic.
The various toxins are commonly associated with particular diseases. For example, toxin type A organisms are associated with myonecrosis (gas gangrene), food-borne illness, and infectious diarrhea in humans, enterotoxemia of lambs, cattle, goats, horses, dogs, alpacas, and other animals; necrotic enteritis in fowl; equine intestinal clostridiosis; acute gastric dilation in non-human primates, and various other animal species, including humans. Toxin type B organisms are associated with lamb dysentery, ovine and caprine enterotoxemia (particularly in Europe and the Middle East), and guinea pig enterotoxemia. Toxin type C organisms are associated with Darmbrand (Germany), and pig-bel (New Guinea), struck in sheep, lamb and pig enterotoxemia, and necrotic enteritis in fowl. Toxin type D organisms are associated with enterotoxemia of sheep, and pulpy kidney disease in lambs. Toxin type E organisms are associated with calf enterotoxemia, lamb dysentery, guinea pig enterotoxemia, and rabbit "iota" enterotoxemia. While C. perfringens type A strains are commonly isolated from soil samples, and is also readily found in intestinal contents in the absence of disease, type B, C, D, and E strains apparently do not survive in soils (ie., these strains are obligate parasites).
The earliest reported outbreaks of food-bome disease associated with C. perfringens was reported by McClung in 1945 (McClung, J. Bacteriol., 50:229-231 [1945]). C. perfringens food-borne illness is frequent, but because the symptoms are usually mild, it is often not reported. However, between 1970 and 1980, 567 C. perfringens food-borne illness outbreaks were confinned in England, and approximately 25 outbreaks are reported annually in the United States (See, Hatheway, supra, at p. 78).
In addition to food-borne illness, evidence for C. perfringens enterotoxin-induced diarrhea in the absence of food has accumulated. Most cases of diarrhea are associated with antimicrobial treatment and elderly patients. Thus, prevention of this disease involves the avoidance of unnecessary antimicrobials and the preferential use of narrow-spectrum antimicrobials (ie., rather than broad-spectrum antimicrobials).
Currently, prompt surgical debridement of contaminated wounds is the most effective means to prevent anaerobic cellulitis and gas gangrene, as antimicrobial therapy alone is insufficient Once a clostridial wound infection has become established, prompt surgical debridement is necessary. In cases of anaerobic cellulitis, wide excision of the affected area and debridement are required, while gas gangrene usually requires complete extirpation of the involved muscle (i.e.,usually amputation of the limb is necessitated).
High doses of penicillin are usually administered, although the emergence of penicillin-resistant strains has resulted in the use of clindamycin, chloramphenicol, and metronidazole. However, strains resistant to tetracycline, chloramphenicol, erythromycin, and clindamycin have been observed. Hyperbaric oxygen (3 atm) in a compression chamber is sometimes used, especially in situations where complete debridement is precluded, such as chest infections.
Polyvalent equine antitoxin prepared against toxic filtrates of four species (C. perfringens, C novyi, C. septicum, and C. histolyticum) has been used in the prophylaxis and treatment of gas gangrene. However, its efficacy was not established and it is no longer available for clinical use (Swartz, supra, at p. 645).
What is needed are compositions and methods suitable for the rapid treatment and prophylaxis of disease due to C. perfringens, as well as other clostridial species associated with histotoxic and/or enterotoxic disease.
C. tetani
Although tetanus has been recognized since ancient times (e.g., the disease was described by Hippocrates), it was not hypothesized to have an infectious agent as its cause until 1867 (See e.g., Hatheway, supra, at p. 75). The strictly toxigenic disease caused by C. tetani is often associated with puncture wounds that do not appear to be serious. The organism is readily isolated from a variety of sources, including soil and the intestinal contents of many animal species (e.g., humans, horses, etc.). Disease results upon the production of toxin by the organism at a site of trauma. The toxin rapidly binds to neural tissue, resulting in the paralysis and spasms characteristic of tetanus. Largely due to the availability of effective toxoids, tetanus is now largely a disease of non-immunized animals, including humans. For example, neonatal tetanus due to contamination of the umbilical stump is very prevalent in some areas of the world. The disease ranges in severity from mild (i.e., good response to drugs and a very low mortality rate) to severe (ie., moderate response to drugs and a 20-40% mortality rate), to very severe (poor response to treatment, and a 50-90% fatality rate). In less developed countries, the mortality rates are approximately 85% for neonatal tetanus, and 50% for other forms of the disease. Neonatal tetanus is almost always severe and is highly fatal. Approximately one half of the cases reported worldwide are neonatal tetanus.
In the U.S., the incidence of tetanus between 1979 and 1986 ranged from between about 60 and 95 cases per year (See, Hatheway, supra, at p. 75); in 1996, there were 36 reported cases (Morbidity and Mortality Weekly Report, Summary of Notifiable Diseases, United States, 1996, 45(53):3, [Oct. 31, 1997]). Worldwide, there are one million cases reported each year (Wells and Wilkins, "Clostridia: Sporeforming Anaerobic Bacilli," in Baron (ed.), Medical Microbiology, 4th ed., University of Texas Medical Branch at Galveston [1991], at p. 268). In some countries, tetanus is still one of the ten leading causes of death.
Tetanus is an extremely dramatic disease resulting from the action of the potent neurotoxin (tetanospasmin). The toxin binds to gangliosides in the central nervous system, and blocks inhibitory impulses to the motor neurons, resulting in prolonged muscle spasms of both flexor and extensor muscles. C. tetani also produces "tetanolysin," an oxygen-sensitive hemolysis that is finctionally and serologically related to streptolysin O, and the oxygen-sensitive hemolysis of various other organisms, including at least six Clostridium species (See e.g., Hatheway, at p. 76). This toxin lyses a variety of cells, including erythrocytes, polymorphonuclear leukocytes, macrophages, fibroblasts, ascites tumor cells, HeLa cells, and platelets. It has an affinity for cholesterol and related sterols. Although in experimental studies, the toxin has been shown to cause pulmonary edema and death in mice, intravascular hemolysis in rabbits and monkeys, and cardiotoxic effects in monkeys, its role in C. tetani infections remains in question (See, Hatheway, at p. 77).
Although the diagnosis of tetanus is relatively easy in advanced cases, successful treatment depends upon early diagnosis before a lethal amount of toxin can become fixed to neural tissue. Thus, patients are usually treated empirically, prior to receiving laboratory data. Tetanus toxoid is used prophylactically to prevent disease. For immunosuppressed patients who may not respond to prophylactic injections of toxoid, human tetanus immunoglobulin given intramuscularly may be used.
Treatment of diagnosed tetanus involves debridement of the wound to remove the organism from the wound site. This debridement occurs after the patient's spasms have been controlled by benzodiazepines. Penicillin or metronidazole is often used to treat the patient, but may not be necessary. However, it has been hypothesized that penicillin may have an adverse effect by acting synergistically with tetanospasmin (Wells and Wilkins, supra, at p. 269-270). Thus, metronidazole is currently recommended. Human tetanus immunoglobulin is also administered intramuscularly. Supportive treatment (e.g., respiratory assistance, nutritional support and intravenous fluids) is often crucial in patient survival. Analgesics that do not cause respiratory depression are also often used (e.g., codeine, meperidine, and morphine). In cases of clean, minor wounds, tetanus toxoid is administered if the patient has not had a booster dose within the past 10 years, although for serious wounds, toxoid is administered if the patient has not had a booster within the past five years.
Summary
In view of the severity and widespread occurrence of clostridial diseases, it is clear that improved methods and compositions to prevent and treat such diseases are needed. Indeed, methods that are suitable for large-scale production of anti-toxin would be useful in various settings, including in less developed countries, where the resources to provide such anti-toxins are limited.